In the present invention, an improvement is made to the clotting end point detector of a known blood clotting time measuring device to facilitate the accurate and consistent detection of the clotting end point to provide a reliable measurement of the blood clotting time.
Measuring blood coagulation or clotting time is necessary for caring for medical patients with tendencies to bleed and for controlling anti-coagulant therapy for thrombotic diseases. Representative techniques employed are Prothrombin Time measurement (PT) for checking the extrinsic coagulation system, Activated Partial Thromboplastine Time measurement (APTT) for the intrinsic system and Partial Thromboplastine Time measurement (PTT).
In the above mentioned PT technique a reagent (such as Simplastin manufactured by Warner-Lambert) is added to a plasma sample separated from the blood. An optical property of the sample is then monitored, such as (1) absorbance, (2) transmittance or optical decay ratio, (3) intensity or logarithm of the light scattered by the sample, (4) refractive index, or (5) the sum or difference of the above going (1), (2), (3) and (4). Hereinafter, the instantaneous value of the monitored optical property or properties will be designated as A. The moment when the detected value of A changes radically is taken as the point where clotting ends, and the time interval from when the reagent is added to when clotting ends is taken as the prothrombin time or PT.
In the APTT technique a reagent (such as Platelin-plus-activator manufactured by Warner-Lambert) is added to a plasma sample, which is reacted at 37.degree. C. for a length of time specified for the reagent (such as for 5 minutes). Then a coagulant agent (such as a solution of calcium chloride) is added. As in the case of the PT technique, one or more optical properties of the sample are monitored, and the point where the value A changes radically is taken as "the clotting end point" and the time interval from when the agent is added to when clotting ends is taken as APTT. PTT is substantially the same as APTT.
The curve for the monitored value of A in respect of one or more optical properties of the plasma sample when a coagulating reagent has been added, changes gradually in the initial stage, changes more rapidly as clotting proceeds, and then finally converges to a certain value.
The difference between the value of A prior to coagulation and after the completion of coagulation varies and depends on the sample. In the case of absorbance, it is approximately 0.01-0.1 (Abs). Blood clotting time is taken by measuring the time from when the coagulating agent is added to when the optical properties of the sample change. Conventionally, two methods are used in detecting the point where the optical properties change or when clotting is completed.
The first method is to differentiate the curve A with respect to time and to detect when this differential dA/dt exceeds a certain predetermined threshold value, this moment being taken as the clotting end point. In this method, when the setting of the threshold value is changed relative to the chronological change in the value of A during clotting, then the moment detected as the clotting end point will also change. Accordingly, if the amplitude of the first differential of curve A changes, the clotting end point also changes. For this reason, threshold values have been set empirically in the prior art. In order to avoid the empirical factors considered undesirable for measuring the clotting time, the following method has been used.
The second method takes as the clotting end point not a predetermined gradient of curve A but the steepest part of this curve A. In other words, curve A is differentiated twice to obtain the second differential d.sup.2 A/dt.sup.2 curve with respect to time. The point where the amplitude of this curve crosses zero from the positive side to the negative side is taken as the clotting end point. In this case, the clotting end point is determined by the optical properties of the sample alone because no threshold value is used and therefore no influence is exerted by the level at which threshold values are chosen. This second method, therefore, is advantageous in that no empirical factors enter in determining the clotting end point.
As has been explained above, the first and the second methods are useful for plasmas showing normal clotting time characteristics. Generally, blood coagulation tests should measure not only the clotting time but should also adjust the said clotting time with the activation curve or the relation of clotting time and coagulation factor concentration. In this case, the activating curve presents the relation between coagulation factor concentrations, or the concentrations of normal pooled plasma in the solution of normal plasma diluted with PSS (physiological saline solution), fiblinogen or adsorped plasma. When normal pooled plasma is diluted 10 times with PSS to seek the above activating curve, the difference between the value of A taken prior to clottoing and after sufficient clotting decreases to about 1/10th of that of the normal plasma. Clotting takes place partially and not uniformly through the sample, and the value A changes intermittently and not uniformly with time, then converging to a certain value. The abnormal plasma which has a longer clotting time also presents the same tendency.
Thus, the curve A for the above samples includes an additional wave component as if a higher frequency noise had been superimposed upon it. When clotting time in respect of such samples was measured by the first method mentioned above, the noise factor emphasized by differentiation caused the first differential curve dA/dt to exceed the threshold value several times so as to generate several signals representing false end points, thus making it difficult to judge the true end point.
As mentioned above, the advantage of the second method is that the clotting end point may be determined only by the optical properties of the sample. However, when the curve A includes a noise factor accompanying the coagulation process, the noise factor is differentiated twice and generates numerous zero crossings of the second differential from positive to negative, making it more difficult than in the first method to determine correctly the clotting end point. This is because its sensitivity toward the high frequency factor of noise is higher in the second differentiation than in the first differentiation.
In the prior art, a low pass filter has been used in an effort to eliminate noise, but the results have not been sufficiently satisfactory.
In one example normal plasma was diluted 10 times with PSS and APTT was checked. After the coagulant agent was added to the plasma, the value a measured in respect of optical properties of the sample gradually decreased with the time contrary to the case of normal plasma, slightly increased near the coagulation end point, and thereafter converged to a certain value.
In this test the second differential curve d.sup.2 A/dt.sup.2 crossed the zero from positive to negative not just once at the true end point but many times.
Thus, when the second method mentioned above is used in testing such a sample, many of signals are generated representing false end points besides the one for the true end point and cause a great deal of errors.